Restoration of Correct Splicing of Thalassemic β-Globin Pre-mRNA by Modified U1 snRNAs

The T-->G mutation at nucleotide 705 in the second intron of the beta-globin gene creates an aberrant 5' splice site and activates a 3' cryptic splice site upstream from the mutation. As a result, the IVS2-705 pre-mRNA is spliced via the aberrant splice sites leading to a deficiency of beta-globin mRNA and protein and to the genetic blood disorder thalassemia. We have shown previously that in cell culture models of thalassemia, aberrant splicing of beta-thalassemic IVS2-705 pre-mRNA was permanently corrected by a modified murine U7 snRNA that incorporated sequences antisense to the splice sites activated by the mutation. To explore the possibility of using other snRNAs as vectors for antisense sequences, U1 snRNA was modified in a similar manner. Replacement of the U1 9-nucleotide 5' splice site recognition sequence with nucleotides complementary to the aberrant 5' splice site failed to correct splicing of IVS2-705 pre-mRNA. In contrast, U1 snRNA targeted to the cryptic 3' splice site was effective. A hybrid with a modified U7 snRNA gene under the control of the U1 promoter and terminator sequences resulted in the highest levels of correction (up to 70%) in transiently and stably transfected target cells

Modification of Alternative Splicing of Bcl-x Pre-mRNA in Prostate and Breast Cancer Cells: ANALYSIS OF APOPTOSIS AND CELL DEATH

There is ample evidence that deregulation of apoptosis results in the development, progression, and/or maintenance of cancer. Since many apoptotic regulatory genes (e.g. bcl-x) code for alternatively spliced protein variants with opposing functions, the manipulation of alternative splicing presents a unique way of regulating the apoptotic response. Here we have targeted oligonucleotides antisense to the 5'-splice site of bcl-x(L), an anti-apoptotic gene that is overexpressed in various cancers, and shifted the splicing pattern of Bcl-x pre-mRNA from Bcl-x(L) to Bcl-x(S), a pro-apoptotic splice variant. This approach induced significant apoptosis in PC-3 prostate cancer cells. In contrast, the same oligonucleotide treatment elicited a much weaker apoptotic response in MCF-7 breast cancer cells. Moreover, although the shift in Bcl-x pre-mRNA splicing inhibited colony formation in both cell lines, this effect was much less pronounced in MCF-7 cells. These differences in responses to oligonucleotide treatment were analyzed in the context of expression of Bcl-x(L), Bcl-x(S), and Bcl-2 proteins. The results indicate that despite the presence of Bcl-x pre-mRNA in a number of cell types, the effects of modification of its splicing by antisense oligonucleotides vary depending on the expression profile of the treated cells

Effects of phenols extracted from olive vegetation water on oxysterols, mutagenicity and genotoxicity of cooked beef hamburgers

By-products of the olive oil mechanical extraction process are sources of high value molecules for the production of new foods and/or ingredients with higher functionality. In particular, replacing chemical additives (i.e. nitrite/nitrate salts) in meat preparations with natural compounds from agro-food by-products (such as biophenols) is of great interest to both food industry and consumers, because it increases the sustainability of the food chain while promoting at the same time the launching of clean label meat products. Due to its high concentration in secoiridoids\u2019 derivatives and verbascoside, the addition of a phenolic extract from olive vegetation water in fresh meat products, is expected to exert an antioxidant action towards lipids and myoglobin, as well as an antimicrobial activity especially versus those Gram-positive bacteria having a significant impact on food safety, such as Listeria monocytogenes and Clostridium botulinum. The aim of this study was to evaluate the effect of an extract rich in phenols obtained from olive vegetation water on the oxidative stability of cooked beef hamburgers prepared without chemical additives during storage, as well as on their mutagenicity and genotoxicity. To prepare the hamburgers, minced beef meat was added with 0.8% salt, 2 starter cultures (SafePro\uae and Bactoferm\uae) and different concentrations of phenolic extract: control (C), L1 (0.35% of spray-dried phenolic extract, equivalent to 87.5 mg of phenols/Kg), L2 (0.70% of spray-dried phenolic extract, equivalent to 175 mg of phenols/Kg). Raw hamburgers were packed under modified atmosphere and stored under alternating exposure to fluorescent light (12 h dark/12 h light) at 4\ub12 \ub0C for 9 days. Hamburgers were sampled at different times (0, 6 and 9 days) and grilled at 70 \ub0C. The oxidative stability of the cooked hamburgers was evaluated by thiobarbituric acid reactive species (TBARs) and oxysterols. To assess the in vitro DNA damage and mutagenicity, the Comet assay and the Ames test were performed on the extract of cooked hamburgers, respectively. The results show that the phenolic extract at both concentrations proved to effectively reduce TBARs and oxysterols during shelf-life. Remarkably, TBARs and oxysterols were up to 5.7- and 4-fold lower in phenol-enriched cooked hamburgers, respectively, as compared to the control samples. Moreover, the extract of cooked hamburgers proved to be genotoxic on PBMCs (Primary Peripheral Blood Mononuclear Cells) humans\u2019 cells, while they were not mutagenic. Nevertheless, the genotoxicity was reduced by presence of the phenolic extract. In conclusion, the phenolic extract from olive oil wastewater proved to be an effective antioxidant and to reduce the production of genotoxic compounds (responsible for carcinogenicity of red meats, according to IARC), thus confirming to be a promising ingredient for clean label fresh meat products

Superoxide-dependent iron uptake a new role for anion exchange protein 2

Lung cells import iron across the plasma membrane as ferrous (F

Unburned Tobacco Cigarette Smoke Alters Rat Ultrastructural Lung Airways and DNA

INTRODUCTION: Recently, the Food and Drug Administration authorized the marketing of IQOS Tobacco Heating System as a Modified Risk Tobacco Product based on an electronic heat-not-burn technology that purports to reduce the risk. METHODS: Sprague-Dawley rats were exposed in a whole-body mode to IQOS aerosol for 4 weeks. We performed the chemical characterization of IQOS mainstream and we studied the ultrastructural changes in trachea and lung parenchyma of rats exposed to IQOS stick mainstream and tissue pro-inflammatory markers. We investigated the reactive oxygen species amount along with the markers of tissue and DNA oxidative damage. Moreover, we tested the putative genotoxicity of IQOS mainstream through Ames and alkaline Comet mutagenicity assays. RESULTS: Here, we identified irritating and carcinogenic compounds including aldehydes and polycyclic aromatic hydrocarbons in the IQOS mainstream as sign of incomplete combustion and degradation of tobacco, that lead to severe remodelling of smaller and largest rat airways. We demonstrated that IQOS mainstream induces lung enzymes that activate carcinogens, increases tissue reactive radical concentration; promotes oxidative DNA breaks and gene level DNA damage; and stimulates mitogen activated protein kinase pathway which is involved in the conventional tobacco smoke-induced cancer progression. CONCLUSIONS: Collectively, our findings reveal that IQOS causes grave lung damage and promotes factors that increase cancer risk. IMPLICATIONS: IQOS has been proposed as a safer alternative to conventional cigarettes, due to depressed concentration of various harmful constituents typical of traditional tobacco smoke. However, its lower health risks to consumers have yet to be determined. Our findings confirm that IQOS mainstream contains pyrolysis and thermogenic degradation by-products, the same harmful constituents of traditional cigarette smoke, and, for the first time, we show that it causes grave lung damage and promotes factors that increase cancer risk in the animal model

Alternative splicing and biological heterogeneity in prostate cancer

The biological diversity of prostate cancer confounds standardization of therapy. Advances in molecular profiling suggest that differences in the genetic composition of tumors significantly contribute to the complexity of the disease. Alternative pre-mrNA splicing is a key genetic process underlying biological diversity. During alternative splicing, coding and noncoding regions of a single gene are rearranged to generate several messenger RNA transcripts yielding distinct protein isoforms with differing biological functions. Misregulation of the splicing machinery and mutations in key regulatory elements affect splicing of cancer-relevant genes. in prostate cancer, aberrant and alternative splicing generates proteins that influence cell phenotypes and survival of patients. splicing events may be exploited for clinical benefit, and technological advances are beginning to uncover novel biomarkers and therapeutic targets. since splicing mediates information transfer from the genome to the proteome, it adds an important dimension to '-omics'-based molecular signatures used to individualize care of patients

Engineering splicing factors with designed specificities

Alternative splicing is generally regulated by trans-acting factors that specifically bind pre-mRNA to activate or inhibit the splicing reaction. This regulation is critical for normal gene expression, and dysregulation of splicing is closely associated with human diseases. Here we engineer artificial splicing factors by combining sequence-specific RNA-binding domains of human Pumilio1 with functional domains that regulate splicing. We applied these factors to modulate different types of alternative splicing in selected targets, examine the activity of effector domains from natural splicing factors, and modulate splicing of an endogenous gene, Bcl-x, an anti-cancer target. The designer factor targeted to Bcl-x increased the pro-apoptotic Bcl-xS splicing isoform, thus promoting apoptosis and increasing chemosensitivity of cancer cells to common anti-tumor drugs. Our approach permits the creation of artificial factors to target virtually any pre-mRNA, providing a new strategy to study splicing regulation and manipulate disease-associated splicing events